Bill C. Bullock

Soy Protein Versus Soy Phytoestrogens in the Prevention of Diet-Induced Coronary Artery Atherosclerosis of Male Cynomolgus Monkeys

Soy protein, long recognized as having cardiovascular benefits, is a rich source of phytoestrogens (isoflavones). To distinguish the relative contributions of the protein moiety versus the alcohol-extractable phytoestrogens for cardiovascular protection, we studied young male cynomolgus macaques fed a moderately atherogenic diet and randomly assigned to three groups. The groups differed only in the source of dietary protein, which was either casein/lactalbumin (casein, n = 27), soy protein with the phytoestrogens intact (soy+, n = 27), or soy protein with the phytoestrogens mostly extracted (soy-, n = 28). The diets were fed for 14 months. Animals fed soy+ had significantly lower total and LDL plus VLDL cholesterol concentrations compared with the other two groups. They soy+ animals had the highest HDL cholesterol concentrations, the casein group had the lowest, and the soy- group was intermediate. A subset was necropsied for atherosclerosis evaluations (n = 11 per group). Morphometric and angiochemical measures were done to quantify atherosclerosis. Coronary artery atherosclerotic lesions were smallest in the soy+ group (90% less coronary atherosclerosis than the casein group and 50% less than the soy- group), largest in the casein group, and intermediate in the soy- group. The effects of the diets on lesion size and arterial lipid measures of the peripheral arteries were similar to those in the coronary arteries, with greatest prevention of atherogenesis with soy+ and intermediate benefit with soy- relative to casein. We could not determine whether the beneficial effects seen in the soy- group relate to the protein itself or to the remaining traces of phytoestrogens. The beneficial effects of soy protein on atherosclerosis appear to be mediated primarily by the phytoestrogen component. Testicular weights were unaffected by the phytoestrogens.

A study of atherosclerosis regression in Macaca mulatta,: IV. Changes in coronary arteries from animals with atherosclerosis induced for 19 months and then regressed for 24 or 48 months at plasma cholesterol concentrations of 300 or 200 mg/dl

Abstract Young adult male rhesus monkeys ( Macaca mulatta ) were fed an atherogenic diet for 19 months. After 19 months of atherosclerosis induction, a baseline group was selected and necropsied to determine the extent and severity of coronary artery atherosclerosis before regression regimens were begun. The remaining animals were fed diets that varied in cholesterol concentration in order to maintain plasma cholesterol concentrations of either about 200 or 300 mg/dl for either 24 or 48 months. The changes in the coronary arteries of these animals were studied by comparing them to the baseline group. During the course of the experiment the lumen areas of the coronary arteries of the animals of both groups increased. Using morphometric techniques to determine the changes in sizes of the artery and changes in intimal areas, we were able to determine the amount of increase in lumen area that was due to growth of the coronary arteries and that due to change in intimal lesions. Among the animals maintained at about 300 mg/dl plasma cholesterol concentration, there was little or no increase in lumen areas that could be accounted for by regression of intimal plaque. In contrast, among the animals maintained at about 200 mg/dl plasma cholesterol concentration, a considerable proportion of the increases in lumen areas was due to regression of intimal plaques. The contribution of plaque regression to increases in lumen areas was dependent both on the size of plaques and time.

Increased circulating levels of an endogenous digoxin-like factor in hypertensive monkeys.

An endogenous, immunoreactive digoxin-like factor (endoxin) was measured in the plasma of nonhuman primates with hypertension. Both normotensive and hypertensive rhesus monkeys had levels of endoxin that significantly correlated with their systolic or diastolic blood pressure. Vervet monkeys with experimentally produced chronic Goldblatt hypertension had significantly elevated endoxin, but not plasma renin. These data suggest that increased plasma endoxin may be a contributing factor in the development of hypertension. (Hypertension 4: 348–354, 1982)

Effect of dietary fish oil on coronary artery and aortic atherosclerosis in African green monkeys.

Studies were carried out for 2.5 to 3 years in adult male African green monkeys (grivet subspecies) fed diets containing 22% of calories as lard or fish oil with 40% of calories as fat and 0.75 mg cholesterol/Kcal to determine if isocaloric substitution of menhaden fish oil for lard affects coronary artery atherosclerosis. The average total plasma cholesterol concentrations during the experimental period were significantly lower for the fish-oil group (231 +/- 37 mg/dl) compared to the lard group (360 +/- 44 mg/dl), but this difference did not become apparent until after 5 months of experimental diet consumption. High density lipoprotein cholesterol concentrations were 30% lower (p less than 0.01) for the fish-oil group also (57 +/- 5 vs. 82 +/- 6 mg/dl). Plasma triglyceride concentrations were low for both groups, but after about 5 months of diet consumption, they were higher for the animals fed fish oil (25 +/- 2 mg/dl) compared to their lard-fed counterparts (15 +/- 1 mg/dl). Coronary artery intimal area (in this case a measure of early atherosclerotic lesion size) was low in all animals but was significantly less (p less than 0.03) for the fish oil vs. lard groups (0.01 +/- 0.002 vs. 0.03 +/- 0.009 mm2). More atherosclerosis was found in other arteries, and a trend was seen of less atherosclerosis in the thoracic aorta and common carotid arteries of the fish-oil group. The size of lesions in the abdominal aorta was similar between diet groups, but microscopic examination of arteries of the lard group revealed relatively more cholesterol monohydrate crystals compared to the arteries of the fish-oil group. Chemical analysis showed that there was less esterified cholesterol (1.46 +/- 0.71 vs. 3.43 +/- 0.74 mg/g, p = 0.04) and free cholesterol (3.7 +/- 2.15 vs. 7.05 +/- 1.68 mg/g, p = 0.08) in the abdominal aortas taken from the animals fed fish oil. There was a significant correlation between low density lipoprotein (LDL) cholesteryl ester (CE) fatty acid ratio (i.e., saturated + monounsaturated/polyunsaturated species) and the amount of esterified (r = 0.59) and free (r = 0.63) cholesterol in the abdominal aortas. Compared to the lard group, animals fed fish oil had significantly lower LDL CE melting temperatures (26 +/- 1 vs. 38 +/- 1 degree C) and significantly smaller LDL particles (2.68 +/- 0.10 vs. 3.25 +/- 0.38 g/mumol). Therefore, the potentially antiatherogenic effects of dietary fish oil include its ability to decrease the concentration, size, CE content, and CE melting temperature of plasma LDL.

Dietary ethanol-induced modifications in hyperlipoproteinemia and atherosclerosis in nonhuman primates (Macaca nemestrina).

MaleMacaca nemestrina were studied In an experiment with a 2 × 2 factorial design. Diets contained low vs high cholesterol levels (0.3 vs 1.0 mg/Kcal) and no ethanol or ethanol, as 36% of the calories substituted isocalorically for carbohydrate. After receiving their diets for 18 months, the monkeys had blood samples drawn for lipoproteln analyses, and then were killed for evaluation of the extent of atherosclerosis. Ethanolfed groups had significantly Increased concentrations of serum cholesterol, triglycerides, low density lipoproteln, and high density lipoprotein. The molecular weight of the low density lipoprotein particles was lower In ethanol-fed animals and the cholesterol esters of low density lipoproteln and high density lipoprotein contained relatively more cholesteryl linoleate and less cholesteryl oleate. Dietary cholesterol had the effect of increasing the concentration of low density lipoprotein (primarily via increasing the low density lipoprotein molecular weight) and decreasing the concentration of high density lipoproteln. Significant Interactions were found between the effects of ethanol and cholesterol on high density lipoprotein and low density lipoprotein. Ethanol significantly decreased the cholesterol-Induced atherosclerosis found in the aorta and coronary arteries. Highly significant correlations between coronary artery atherosclerosis and low density lipoproteln molecular weight, Inverse high density lipoproteln concentration, and low density lipoprotein cholesterol ester pattern were found. In contrast, low density lipoproteln molar concentration (number of low density lipoproteln particles per liter of plasma) was not significantly correlated with coronary artery atherosclerosis. Different relationships with aortic atherosclerosis were found; low density lipoproteln molecular weight and cholesterol ester pattern were highly correlated, while high density lipoproteln concentration was not. The high correlations found between lipoproteln characteristics and atherosclerosis severity suggest that the effect of ethanol in reducing the development of atherosclerosis may have been mediated through Its effects on the plasma llpoprotelns.

Accelerated onset of uterine tumors in transgenic mice with aberrant expression of the estrogen receptor after neonatal exposure to diethylstilbestrol

The role of estrogen and the estrogen receptor (ER) in the induction and promotion of tumors was investigated by using transgenic MT‐mER mice, which overexpress the ER. It was hypothesized that because of this abnormal expression of the ER, the reproductive‐tract tissues of the MT‐mER mice may be more susceptible to tumors after neonatal exposure to the potent synthetic estrogen diethylstilbestrol (DES). Normally non–estrogen responsive tissues that may have expressed ER as a result of the transgene were also studied for DES‐induced tumors. Wild‐type and MT‐mER littermates were treated with 2 μg/pup/d DES 1–5 d after birth and then killed at 4, 8, 12, and 18 mo of age. The DES‐treated MT‐mER mice demonstrated a significantly higher incidence of uterine adenocarcinoma at 8 mo (73%) than the DES‐treated wild‐type mice (46%). The tumors of the MT‐mER mice were often more aggressive than those in the wild‐type animals. These tumors were also preceeded at 4 mo by a significantly higher incidence of the preneoplastic lesion atypical hyperplasia in the MT‐mER mice (26% compared with 0% in the wild‐type mice). Other DES‐induced abnormalities were observed at equal rates in the wild‐type and MT‐mER mice. Although no tumors were observed in untreated wild‐type females, a single untreated MT‐mER female had uterine adenocarcinoma at 18 mo. These data indicate that the level of ER present in a tissue may also be a determining factor in development of estrogen‐responsive tumors. Mol. Carcinog. 19:236–242, 1997.

Dietary polyunsaturated fat decreases coronary artery atherosclerosis in a pediatric-aged population of African green monkeys.

The hypothesis tested was that juvenile African green monkeys consuming diets enriched with n-6 polyunsaturated fat from birth until young adulthood would have significantly less coronary artery atherosclerosis than comparable animals consuming diets enriched with saturated fat. African green monkeys (Cercopithecus aethiops, n = 108) of both sexes were fed atherogenic diets (0.8 mg cholesterol/kcal) throughout their lives so that death at 16, 32, or 60 months of age permitted quantification of atherosclerosis. In the coronary arteries, the average intimal area increased significantly with age (P = .02), showing increases of 28-fold and sevenfold between 32 and 60 months in the saturated fat- and polyunsaturated fat-fed groups, respectively. Young adult male animals at 60 months of age were found to have significantly (P = .03) more coronary artery atherosclerosis than female animals. Animals fed polyunsaturated fat had significantly (P < or = .01) less coronary artery atherosclerosis. By 60 months of age in the animals consuming polyunsaturated fat, the average coronary artery intimal area was one fourth and the average size of the largest coronary intimal lesion was one fifth that in monkeys fed saturated fat. Low-density lipoprotein (LDL) cholesterol and LDL particle size were each found to be positively correlated with coronary artery atherosclerosis end points in both diet groups. In addition to the coronary arteries, atherosclerosis in the abdominal and thoracic aorta and carotid arteries was also evaluated; the coronary arteries were the only arterial system with significantly less atherosclerosis in the polyunsaturated fat group as measured by intimal area. However, evaluation of histological sections of abdominal aorta showed relatively more sterol clefts in the saturated fat-fed group, and more free cholesterol was measured, suggesting that lesions were more complicated in this group. These results show that dietary intervention early in life with n-6 polyunsaturated fat can be effective in decreasing the development of atherosclerosis, particularly in the coronary arteries of primates. This outcome supports the concept that dietary intervention beginning early in childhood can have beneficial effects on the coronary heart disease of later life.

Naturally Occurring and Experimental Diabetes in Cynomolgus Monkeys: A Comparison of Carbohydrate and Lipid Metabolism and Islet Pathology

Diabetes is a major health problem of increasing incidence in the United States. Diabetes research has been limited by lack of availability of good animal models, particularly for the study of comorbidities associated with diabetes. We investigated the use of cynomolgus monkeys as an animal model of both type 1 and type 2 diabetes and compared these naturally occurring diseases with streptozotocin-induced diabetes. Both type 1 diabetics and streptozotocin-induced diabetics present with sudden onset of hyperglycemia and are ketosis prone without exogenous insulin. Type 2 diabetics can have a very long period of moderate hyperglycemia and hypertriglyceridemia and only require exogenous insulin therapy if pancreatic islet reserves are depleted. Type 2 diabetes is preceded by a relatively long period of insulin resistance that is associated with obesity and dyslipidemia. As insulin resistance progresses, islet size and insulin content increases initially. However, with sustained periods of insulin resistance, islet amyloid polypeptide (IAPP) is deposited in islets and can replace normal islet architecture, resulting in an insulin-deficient state. Appearance of IAPP also occurs in human type 2 diabetics but not in conventional rodent models. Unlike type 2 diabetes, neither type 1 nor streptozotocin-induced diabetes is associated with IAPP. Rather, islets can appear normal histologically, but have decreased insulin secretion and immunostaining. Further, the amount of insulin present in the islet is correlated with plasma insulin levels following glucose challenge. Studies are ongoing to determine the pathogenic changes associated with the progression of diabetes and to find novel drug treatments for diabetics.

Comparative primate atherosclerosis: I. Tissue Cholesterol Concentration and Pathologic Anatomy

Abstract Stump-tailed macaques ( Macaca arctoides ), African green monkeys ( Cercopithecus aethiops ), squirrel monkeys ( Saimiri sciureus ), and woolly monkeys ( Lagothrix lagothricha ) were fed control, solid atherogenic (1 mg cholesterol/cal) or liquid diets containing 0, 0.5, or 1 mg cholesterol/cal. Stump-tailed macaques fed the solid atherogenic diet had the highest tissue and serum cholesterol concentration (about 700 mg/dl) and the most extensive atherosclerosis. These monkeys appeared to respond differently to diets containing 1 mg cholesterol/cal. Those animals fed the liquid diet had higher liver cholesterol concentration but lower serum cholesterol concentration than animals fed the solid diet. African green monkeys fed the solid atherogenic diet had serum cholesterol concentrations of about 450 mg/dl. A greater percentage of the abdominal aorta was covered by plaque than the thoracic aorta. Coronary artery atherosclerosis was focal with the largest plaques being found in the left main coronary artery. The microscopic appearance of these plaques was similar to that of plaques from people. Squirrel monkeys fed the atherogenic diet were the most variable group. The average serum cholesterol concentration averaged about 450 mg/dl (range: 291 to 716). The percentage of aorta covered by plaque ranged from 0 to 55% with more thoracic than abdominal aortic atherosclerosis. There were findings consistent with hemorrhage in plaques from two animals. These monkeys, like stump-tailed macaques but unlike African green monkeys had relatively high liver cholesterol concentrations. Woolly monkeys appeared to develop atherosclerosis when fed 1 mg cholesterol/cal but did not have greatly elevated serum cholesterol concentrations.

A study of atherosclerosis regression in Macaca mulatta. V. Changes in abdominal aorta and carotid and coronary arteries from animals with atherosclerosis induced for 38 months and then regressed for 24 or 48 months at plasma cholesterol concentrations of 300 or 200 mg/dl.

The influence of two levels of plasma cholesterol concentration on the long-term regression of atherosclerotic plaques in rhesus monkeys (Macaca mulatta) was studied. Forty-eight young adult male rhesus monkeys were fed an atherogenic diet for 19 months, then diets designed to maintain plasma cholesterol concentrations at 280-320 mg/dl (actual 316 +/- 10 mg/dl; mean +/- SEM) of 180-220 mg/dl (actual 204 +/- 4 mg/dl). Twelve animals were killed after 19 months to evaluate the atherosclerosis produced. The remaining 36 monkeys were studied after 48 months of atherosclerosis regression. Significantly greater amounts of accumulated nonesterififed and esterified cholesterol were lost from the arteries of monkeys that underwent regression at about 200 mg/dl, in comparison with 300 mg/dl. Regression at both plasma cholesterol concentrations resulted in increased collagen and decreased elastin content in the thoracic aorta but not in the other arteries studied. After regression at 300 mg/dl there was no change (83%) in the frequency of calcification of the thoracic aorta, while at 200 mg/dl the frequency of calcification was reduced to 50%. When compared with monkeys whose athersclerotic lesions were produced in an identical manner but were regressed for only 24 months, after 48 months had increased cholesterol concenrations in the thoracic aorta and carotid artery but not the abdominal aorta of iliaco-femoral artery, regardless of the plasma cholesterol concentration. The lipid composition of the regressed plaque suggested a change in plaque lipid toward the character of extracellular deposits described physically as crystalline in nature. The physical state was influenced by the length of the regression period and the plasma cholesterol concentration during regeression. In the thoracic aorta, principally as a result of changes in elastin content, the collagen-to-elastin ratio increased between 24 and 48 months of regression. It is proposed that the differences in removal of cholesterol from the thoracic and abdominal aorta after 24 and 48 months of regression in animals at 300 mg/dl may be influenced by the rearrangement of connective tissue. On the basis of the lipid and mineral content of uncomplicated atherosclerotic plaques after 4 yers of regression, it appears that the plasma cholesterol concentration during the regression period is a signigicant factor influencing the extent of plaque regression as well as the potential for further progression.